Our long term objectives are to understand the mechanisms by which developing leaves produce the spatial patterns of gene expression essential for their later function. Our studies of the expression of C4 pathway genes during maize leaf development have indicated that each vein is an early organizing center for expression in surrounding cells. Different C4 genes are expressed proximal and distal to the vein, in a spatial pattern that eventually restricts expression to bundle sheath (BS) and mesophyll (M) cells at near and far locations, respectively. Homologs of the C4 genes are expressed non-specifically in C3 plants. This proposal seeks to determine the molecular basis of these phenomena, with the following specific aims: (1) to measure the dependence of C4 gene expression on cell maturity and cell position, using C4-reporter assays in leaves with various cell ages and arrangements, (2) to characterize the regulatory elements required for cell-specific and position-dependent expression of genes for NADP-dependent malic enzyme (ME, BS-specific) and NADP-dependent malate dehydrogenase (MDH, M-specific), using expression assays in protoplasts and intact leaves, and (3) to determine whether the C4 genes differ from their C3 counterparts by the presence of spatial response elements or whether C4 and C3 anatomies provide different spatial signals to fundamentally similar genes. These studies will be aided by our experience with in situ analysis of gene expression in leaves, by our extensive collection of characterized C4 antibody, cDNA, and gene probes, and by natural differences in leaf cell arrangements among maize, Flaveria, and Arundinella C4 and C4-like species.